A key issue in understanding the proton pumping mechanism of bacteriorhodopsin relates to the manner in which the protein maintains the vectoriality of proton translocation. A proton is transferred from the retinal Schiff base nitrogen to Asp85 on the extracellular side of the channel and another proton is transferred to the Schiff base from Asp96 on the cytoplasmic end of the channel. The gating is assumed to be accomplished during the deprotonated M states through structural changes in the retinal and/or protein which alter the pKa of the Schiff base nitrogen with respect to Asp85 and Asp96. While many of the steps in the photocycle are in equilibrium, detailed analysis of time-resolved visible spectra provides indirect evidence for an irreversible M, _> M2 reaction. Previous work in this laboratory has demonstrated our ability to trap and observe two distinct M intermediates, which occur successively in the photocycle. Investigation of the differences between these two states should reveal the gating mechanism. Changes in the pK, of the Schiff base nitrogen can be accounted for by movement with respect to the proton donor and acceptor. Since the ionone ring is fixed by the tight packing of several residues, the position of the Schiff base nitrogen can be determined from the twisting of the polyene chain. Evidence of a twisted chain comes from FTIR data and molecular dynamics simulations, which suggest that the twist is distributed across multiple bonds. Experimental measurements correlating C-H and/or N-H dipole vectors of directly and indirectly bonded nuclei in the Schiff base polyene chain of various intermediates are currently underway. Correlation of dipole vectors from directly bonded nuclei produces the torsion angle between the two. Correlation of vectors from non-neighboring nuclei depends on all of the torsion angles of each bond between the nuclei, which gives long-range twisting parameters and provides a way to skip past nuclei without a directly attached proton, such as C-13 and C-10 in the retinal chain.